Neuroprotective effects of aucubin on hydrogen peroxide-induced toxicity in human neuroblastoma SH-SY5Y cells via the Nrf2/HO-1 pathway.

BACKGROUND: When redox balance is lost in the brain, oxidative stress can cause serious damage that leads to neuronal loss, in congruence with neurodegenerative diseases. Aucubin (AU) is an iridoid glycoside and that is one of the active constituents of Eucommia ulmoides, has many pharmacological effects such as anti-inflammation, anti-liver fibrosis, and anti-atherosclerosis. PURPOSE: The present study aimed to evaluate the inhibitory effects of AU on cell oxidative stress against hydrogen peroxide (H2O2)-induced injury in SH-SY5Y cells in vitro. METHODS: SH-SY5Y cells were simultaneously treated with AU and H2O2 for 24 h. Cell viability was measured by CCK-8. Additionally, mitochondrial membrane depolarization, reactive oxygen species (ROS) generation, and cell apoptosis were measured by flow cytometry. RESULTS: The results showed that AU can significantly increase the H2O2-induced cell viability and the mitochondrial membrane potential, decrease the ROS generation, malondialdehyde (MDA), and increase glutathione (GSH) contents and the superoxide dismutase (SOD) activity. We also found that H2O2 stimulated the production of nitric oxide (NO), which could be reduced by treatment with AU through inhibiting the inducible nitric oxide synthase (iNOS) protein expression. In H2O2-induced SH-SY5Y cells, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) content and cell apoptosis were significantly reduced by AU treatment through nuclear factor E2-related factor 2/hemo oxygenase-1 (Nrf2/HO-1) activation, inhibiting the expression of p-NF-κB/NF-κB and down-regulating MAPK and Bcl-2/Bax pathways. CONCLUSION: These results indicate that AU can reduce inflammation and oxidative stress through the NF-κB, Nrf2/HO-1, and MAPK pathways.

Ameliorative effects of nano-elemental selenium against hexavalent chromium-induced apoptosis in broiler liver.

The current study examined the ameliorative effects of nano-elemental selenium (Nano-Se) against chromium-VI (K2Cr2O7)-induced apoptosis in chickens. The expression of apoptosis-related genes was evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. A total of 60, one-day-old broiler chickens allotted to six equal groups, i.e., control group (standard diet), Cr(VI)-exposed group (K2Cr2O7 via drinking water), Nano-Se group (Nano-Se at 0.5 mg/kg via diet), protection group (K2Cr2O7 + Nano-Se), cure group (K2Cr2O7 for initial 2 weeks and then Nano-Se), and prevention group (opposite to the cure group) and were detected by the activities of pro-apoptosis (Bax, Caspase-3) and anti-apoptosis (Bcl-2) genes expression at day 35 of the experiment. Intense apoptosis was observed in liver tissues of chickens exposed to K2Cr2O7. The Nano-Se supplementation caused a significant decrease (P < 0.01) in the mRNA expression levels of Bax and Caspase-3 genes, while significantly elevated (P < 0.05) mRNA expression level of Bcl-2 gene was observed in Nano-Se experimental groups as compare to control and Cr(VI)-exposed group. The results quantified by the RT-qPCR were further confirmed by the western blot analysis. Altogether, these results suggest anti-apoptotic effects of Nano-Se in the chicken liver, which is interesting for further study. The present findings suggested that Nano-Se has protective effects against K2Cr2O7-induced apoptosis in broilers liver and can serve a key role as a protective agent against apoptosis.

Cytotoxic and apoptotic potential of Myristica fragrans Houtt. (mace) extract on human oral epidermal carcinoma KB cell lines

Several studies have revealed that certain naturally occurring medicinal plants inhibit the growth of various cancers. The present study was conducted to evaluate cytotoxicity and apoptotic induction potential of Myristica fragrans Houtt mace extract. The cytotoxic activity of the Myristica fragrans Houtt mace acetone extract was assayed by MTT assay on human oral epidermal carcinoma KB cell lines. KB cells were incubated with different concentration of mace extract ranging from 25 to 125 µg/mL for 24hrs. The apoptotic induction potential was also studied by the analysis of Bcl-2 protein and gene expression in mace extract incubated KB cell lines using western blotting technique and real-time polymerase chain reaction. The mace extract exhibited cytotoxicity and anticancer effect against KB cell lines and it also suppressed the growth of cancer cells, therefore growth inhibitory effect was noted in extract treated cell lines. The apoptotic potential of mace extract was accompanied by reduced gene expression of Bcl-2 compared to the untreated KB cells. The mace extract shows the cytotoxic activity and induced the apoptosis through the modulation of its target genes Bcl-2 in the KB cell lines, suggesting the potential of mace as a candidate for oral cancer chemoprevention. This can be further investigated in vivo for its anticancer potential.

Electroacupuncture improves neurobehavioral function and brain injury in rat model of intracerebral hemorrhage.

Acupuncture has been widely used as a treatment for stroke in China for a long time. Recently, studies have demonstrated that electroacupuncture (EA) can accelerate intracerebral hemorrhage (ICH)-induced angiogenesis in rats. In the present study, we investigated the effect of EA on neurobehavioral function and brain injury in ICH rats. ICH was induced by stereotactic injection of collagenase type I and heparin into the right caudate putamen. Adult ICH rats were randomly divided into the following three groups: model control group (MC), EA at non-acupoint points group (non-acupoint EA) and EA at Baihui and Dazhui acupoints group (EA). The neurobehavioral deficits of ICH rats were assessed by modified neurological severity score (mNSS) and gait analysis. The hemorrhage volume and glucose metabolism of hemorrhagic foci were detected by PET/CT. The expression levels of MBP, NSE and S100-B proteins in serum were tested by ELISA. The histopathological features were examined by haematoxylin-eosin (H&E) staining. Apoptosis-associated proteins in the perihematomal region were observed by immunohistochemistry. EA treatment significantly promoted the recovery of neurobehavioral function in ICH rats. Hemorrhage volume reduced in EA group at day 14 when compared with MC and non-acupoint EA groups. ELISA showed that the levels of MBP, NSE and S100-B in serum were all down-regulated by EA treatment. The brain tissue of ICH rat in the EA group was more intact and compact than that in the MC and non-acupoint groups. In the perihematomal regions, the expression of Bcl-2 protein increased and expressions of Caspase-3 and Bax proteins decreased in the EA group vs MC and non-acupoint EA groups. Our data suggest that EA treatment can improve neurobehavioral function and brain injury, which were likely connected with the absorption of hematoma and regulation of apoptosis-related proteins.

Boschniakia Rossica Polysaccharide Triggers Laryngeal Carcinoma Cell Apoptosis by Regulating Expression of Bcl-2, Caspase-3, and P53.

BACKGROUND Laryngeal cancer is a malignant head and neck tumor with high morbidity and high mortality in humans. Recently, treatments with Chinese medicines and their extracts have gradually received great attention, and studies suggest that Boschniakia rossica polysaccharide (BRP) has potential anti-tumor activity. Therefore, this study investigating the role of BRP in inducing apoptosis in human laryngeal carcinoma cells. MATERIAL AND METHODS The BRP was extracted with organic solvent and HR column. We treated Hep2 laryngeal carcinoma cells with different concentrations of BRP, then assessed cell growth inhibition rate by flow cytometry and apoptosis index by TUNEL staining. The protein expression of p53, Bcl-2, Bax, and caspase-3 were analyzed by Western blot. RESULTS Flow cytometry results showed that BRP inhibited Hep2 cell proliferation in a dose-dependent manner (p<0.05), and TUNEL staining indicated that BRP significantly increased Hep2 apoptosis index (p<0.05). Western blot results showed that the expression levels of p53 and activation of caspase-3 in Hep2 cells were significantly up-regulated (p<0.05), while the expression of Bcl-2 was significantly down-regulated (p<0.05). CONCLUSIONS BRP might induce cell apoptosis by regulating the expression level of cell apoptosis-associated proteins, suggesting strong anti-laryngeal cancer activity.

Sanguisorbae radix protects against 6-hydroxydopamine-induced neurotoxicity by regulating NADPH oxidase and NF-E2-related factor-2/heme oxygenase-1 expressions.

6-Hydroxydopamine (6-OHDA) produces neuronal cell damage by generating reactive oxygen species (ROS). The major mechanisms of protection against ROS-induced stress are inhibiting expression of ROS generating genes such as NADPH oxidase (NOX) and increasing expression of endogenous antioxidant genes such as heme oxygenase-1 (HO-1). This study investigated whether a standardized Sanguisorbae Radix extract (SRE), a medical herb commonly used in Asian traditional medicine, has a protective effect on 6-OHDA-induced cell toxicity by regulating ROS in SH-SY5Y cells. SRE at 10 and 50 µg/mL significantly reduced 6-OHDA-induced cell damage dose dependently in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and by Hoechst 33342 staining. SRE increased the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X ratio and decreased cytochrome C release and caspase-3 activity. SRE also abolished 6-OHDA-induced ROS by inhibiting NOX expression and by inducing HO-1 expression via NF-E2-related factor-2 activation. Taken together, these results demonstrate that SRE has protective effects against 6-OHDA-induced cell death by regulating ROS in SH-SY5Y cells.

Dynamin inhibitors induce caspase-mediated apoptosis following cytokinesis failure in human cancer cells and this is blocked by Bcl-2 overexpression.

BACKGROUND: The aim of both classical (e.g. taxol) and targeted anti-mitotic agents (e.g. Aurora kinase inhibitors) is to disrupt the mitotic spindle. Such compounds are currently used in the clinic and/or are being tested in clinical trials for cancer treatment. We recently reported a new class of targeted anti-mitotic compounds that do not disrupt the mitotic spindle, but exclusively block completion of cytokinesis. This new class includes MiTMAB and OcTMAB (MiTMABs), which are potent inhibitors of the endocytic protein, dynamin. Like other anti-mitotics, MiTMABs are highly cytotoxic and possess anti-proliferative properties, which appear to be selective for cancer cells. The cellular response following cytokinesis failure and the mechanistic pathway involved is unknown. RESULTS: We show that MiTMABs induce cell death specifically following cytokinesis failure via the intrinsic apoptotic pathway. This involves cleavage of caspase-8, -9, -3 and PARP, DNA fragmentation and membrane blebbing. Apoptosis was blocked by the pan-caspase inhibitor, ZVAD, and in HeLa cells stably expressing the anti-apoptotic protein, Bcl-2. This resulted in an accumulation of polyploid cells. Caspases were not cleaved in MiTMAB-treated cells that did not enter mitosis. This is consistent with the model that apoptosis induced by MiTMABs occurs exclusively following cytokinesis failure. Cytokinesis failure induced by cytochalasin B also resulted in apoptosis, suggesting that disruption of this process is generally toxic to cells. CONCLUSION: Collectively, these data indicate that MiTMAB-induced apoptosis is dependent on both polyploidization and specific intracellular signalling components. This suggests that dynamin and potentially other cytokinesis factors are novel targets for development of cancer therapeutics.

Sorafenib induces apoptosis and autophagy in prostate cancer cells in vitro.

The multiple tyrosine kinase inhibitor sorafenib has recently demonstrated clinical effects in patients with androgen-independent prostate cancer. These observations provided the rational for investigating the anti-tumoral properties of this compound on prostate cancer cell lines at the molecular level. Two hormone refractory (PC3 and DU145) and one hormone responsive cell line (22Rv1) were used. By use of a panel of cell biology techniques such as immunoblotting, flow cytometry and immunocytochemistry, effects on the MAPK pathway and induction of apoptosis and autophagy were evaluated. We demonstrate that sorafenib reduced cell viability in a dose-dependent manner, induced apoptosis and inactivated the MAPK pathway. Moreover, we show for the first time, that sorafenib treatment of prostate cancer cells also induces cellular autophagy. This feature is in accordance with the anticancer potential of sorafenib and adds another important effector mechanism of this compound. These observations may open potentially interesting treatment combinations that may augment the effect of sorafenib, either by drugs that promote autophagy such as the rapalogues, or by combining sorafenib with compounds that specifically inhibit the autophagic process.

[Study of introduction apoptosis of hepatocarcinoma cell SMMC-7721 by anthraquinone extracted from rubiqmnone and molecular mechanism].

OBJECTIVE: To research the effect of anthraquinone extracted from rubiqmnone on growth inhibition, introduction apoptosis and expression of relative gene of bcl-2 of hepatocarcinoma cell SMMC 7721, and provide the effective target of gene therapy. METHOD: The growth inhibition effect was detected by MTF. Morpholgy, DNA agarose gel electrophoresis and flow cytometry were used to observe the cell apoptosis. The effect of anthraquinone on bcl-2mRNA expression was analyzed by RT-PCR. RESULT: Anthraquinone could inhibit the growth of hepatocarcinoma cell SMMC 7721. The typical apoptosis cells were found by inverted microscope and common microscope. DNA agarose gel electrophoresis showed a typical apoptosis strip. G1 period of cell cycle had apoptosis peak of abnormal diploid by PI simple stain, and cell cycle stopped at G1 period. The apoptosis fuction of anthraquinone introdution hepatocarcinoma cell was further certified by Annexin V-FITC/PI. Anthraquinone could decrease the expression of bcl-2mRNA by RT-PCR. CONCLUSION: Anthraquinone can significantly inhibit growth of hepatocarcinoma cell and induce apoptosis. The mocular mechanism may be related to down-regulating the expression of bcl-2 gene.

Achyranthes bidentata Blume extract protects cultured hippocampal neurons against glutamate-induced neurotoxicity.

UNLABELLED: We have prepared an aqueous extract of Achyranthes bidentata Blume, a Chinese medicinal herb commonly prescribed for arthritis treatment or immnopotentiation, and have found that Achyranthes bidentata extract promotes nerve growth and prevents neuronal apoptosis. AIM OF THE STUDY: To investigate the protective effect of Achyranthes bidentata extract against glutamate-induced neurotoxicity in primary culture of rat hippocampal neurons. MATERIALS AND METHODS: We accomplished MTT assay for cell viability, Hoechst 33342 staining, and flow cytometry for cell apoptosis analysis to examine the effects of Achyranthes bidentata extract on glutamate-induced neurotoxicity, and also used Fluo 4-AM measurement, RT-PCR and Western blot analysis to determine the changes in intracellular calcium concentration [Ca(2+)](I), and mRNA and protein levels of Bcl-2, respectively, concurrently accompanied with the influences of Achyranthes bidentata extract. RESULTS: Achyranthes bidentata extract was found to inhibit glutamate-induced neuronal damage in a dose- and time-dependent manner. On the other hand, Achyranthes bidentata extract depressed glutamate-induced elevation of intracellular calcium concentration [Ca(2+)](i), and also antagonized glutamate-evoked decreases in Bcl-2 expression at mRNA and protein levels. CONCLUSION: The results suggest that Achyranthes bidentata extract prevents glutamate-induced cell damage in primarily cultured hippocampal neurons by inhibiting an increase in [Ca(2+)](i), and reversing the down-regulation of Bcl-2.

Effect of a seashell protein Haishengsu on cell growth and expression of apoptosis genes in leukemia K562 cells.

OBJECTIVES: To investigate the effect of a seashell protein Haishengsu (HSS), an extract from a shellfish Tegillarca granosa, on cell growth and the expression of apoptosis genes in leukemia K562 cells. METHODS: Cultured K562 cells were treated with HSS at various concentrations (10-40 mg/L). The cell cycle, cell growth and the expression of apoptosis suppressor gene bcl-2 and apoptosis promoting gene bax were evaluated. RESULTS: HSS, 20mg/L, inhibited cell cycle in the G0/G1 and S phases. HSS, 20mg/L, also inhibited the growth of K562 cells over time. Expression of bcl-2 gene in the HSS 20mg/L (58.8%+/-4.7%) and HSS 40 mg/L group (26.6%+/-2.1%) were lower than in the control group (91.0+/-8.7%, P < 0.01). Expression of bax gene in the HSS 20mg/L (77.7+/-3.6%) and 40 mg/L group (90.6+/-3.7%) were higher than in the control group (10.9+/-6.6%, P < 0.01). CONCLUSION: HSS suppresses leukemia K562 cell growth by inhibiting the G0/G1 and S phases of the cell cycle. It also induces apoptosis in these leukemia cells by reducing the expression of apoptosis suppressor gene bcl-2, and increasing the expression of apoptosis promoting gene bax. Further studies are required to investigate the clinical efficacy of HSS in leukemia.

Intrauterine growth restriction due to uteroplacental vascular insufficiency leads to increased hypoxia-induced cerebral apoptosis in newborn piglets.

Uteroplacental vascular insufficiency in humans is a common cause of intrauterine growth restriction (IUGR) and is associated with an increased incidence of perinatal asphyxia and neurodevelopmental disorders compared to normal weight newborns. Experimental models that provide an opportunity to analyze the pathogenesis of these relationships are limited. Here, we used neonatal pigs from large litters in which there were piglets of normal birth weight (for controls) and of low birth weight (for uteroplacental vascular insufficiency). Hypoxia was induced in paired littermates by reducing the fraction of inspired oxygen to 4% for 25 min. Brain tissue was collected 4 h post-hypoxia. Cerebral levels of apoptosis were quantified morphologically and verified with caspase-3 activity and TUNEL. Expression of Bcl-2, Bcl-XL and Bax proteins was investigated using immunohistochemistry. Cellular positivity for Bcl-2 was consistently higher in the non-apoptotic white matter of the hypoxic IUGR animals compared with their littermates and reached significance at P < 0.05 in several pairs of littermates. Alterations in Bax showed a trend towards higher expression in the hypoxic IUGR littermates but rarely reached significance. The IUGR piglets showed a significantly greater amount of apoptosis in response to the hypoxia than the normal weight piglets, suggesting an increased vulnerability to apoptosis in the IUGR piglets.

Propofol dose-dependently reduces tumor necrosis factor-alpha-Induced human umbilical vein endothelial cell apoptosis: effects on Bcl-2 and Bax expression and nitric oxide generation.

We investigated whether propofol can inhibit tumor necrosis factor (TNF)-alpha-induced apoptosis in cultured human umbilical vein endothelial cells (HUVECs). Isolated HUVECs were cultured in Dulbecco's modified Eagle medium supplemented with 20% bovine calf serum. HUVECs in untreated and propofol control groups were cultured at 37 degrees C for 24.5 h. HUVECs in the TNF treatment groups were initially cultured for 30 min in the presence of TNF or various concentrations of propofol, respectively, which were then cultured for 24 h with the addition of TNF at 40 ng/mL in the medium. Apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and confirmed by electron microscopy. The antiapoptotic Bcl-2 and proapoptotic Bax protein expressions were measured by immunocytochemical analysis. TNF stimulation resulted in a reduced Bcl-2/Bax ratio and increased apoptotic index (AI: percentage of apoptotic cells) in HUVECs. Propofol, at concentrations >/=12 muM, significantly (P < 0.001) and dose-dependently attenuated TNF-induced increase in AI and decrease in Bcl-2/Bax ratio. This was accompanied by increases in nitric oxide production. There is an inverse correlation between the ratio of Bcl-2/Bax expression and AI (P = 0.0009). These results suggest that propofol, at clinical relevant concentrations, can reduce TNF-induced HUVEC apoptosis.

Alternative splicing of Bcl-2-related genes: functional consequences and potential therapeutic applications.

Apoptosis is a morphologically distinct form of cell death. It is executed and regulated by several groups of proteins. Bcl-2 family proteins are the main regulators of the apoptotic process acting either to inhibit or promote it. More than 20 members of the family have been identified so far and most have two or more isoforms. Alternative splicing is one of the major mechanisms providing proteomic complexity and functional diversification of the Bcl-2 family proteins. Pro- and anti-apoptotic Bcl-2 family members should function in harmony for the regulation of the apoptosis machinery, and their relative levels are critical for cell fate. Any mechanism breaking down this harmony by changing the relative levels of these antagonistic proteins could contribute to many diseases, including cancer and neurodegenerative disorders. Recent studies have shown that manipulation of the alternative splicing mechanisms could provide an opportunity to restore the proper balance of these regulator proteins. This review summarises current knowledge on the alternative splicing products of Bcl-2-related genes and modulation of splicing mechanisms as a potential therapeutic approach.

Overexpression of heme oxygenase (HO)-1 renders Jurkat T cells resistant to fas-mediated apoptosis: involvement of iron released by HO-1.

We recently demonstrated that heme oxygenase (HO)-1 is constitutively expressed in human CD4+CD25+ regulatory T cells and induced by anti-CD28 or anti-CD28/anti-CD3 stimulation, even in CD4+CD25- responder T cells. To study the effects of HO-1 expression on lymphocyte survival, we transfected the HO-1 gene or induced the gene to express HO-1 protein with cobalt protoporphyrin (CoPP) in Jurkat T cells. Consistently, anti-Fas antibody triggered apoptotic cell death in wild-type Jurkat T cells. Surprisingly, however, HO-1-overexpressing Jurkat T cells showed strong resistance to Fas-mediated apoptosis. In contrast, abrogation of HO-1 expression by antisense oligomer against HO-1 gene from CoPP-treated cells or depletion of iron by desferrioxamine from HO-1-transfected cells abolished the resistance. In addition, exogenously added iron rendered wild-type Jurkat T cells resistant. The resistance involved IkappaB kinase (IKK) activation via iron-induced reactive oxygen species formation, NF-kappaB activation by activated IKK, and c-FLIP expression by activated NF-kappaB. Primary CD4+ T cells induced by CoPP to express HO-1 also showed more resistance to Fas-mediated apoptosis than untreated cells. Our findings suggest that HO-1 plays a critical and nonredundant role in Fas-mediated activation-induced cell death of T lymphocytes.

Prognostic significance of p53, bcl-2, and Bax expression in early breast cancer.

BACKGROUND: The use of adjuvant chemotherapy in early breast carcinoma is controversial, with most advocating its use in high-risk patients as defined by specific clinicopathologic parameters. Both bcl-2 and p53, which play a role in determining tumor growth by their effects on apoptosis and cell proliferation respectively, may serve to delineate this subset more accurately. The purpose of this study was to determine the prognostic value of bcl-2, Bax, and mutant p53 in stage I breast cancer. STUDY DESIGN: A total of 75 patients with stage Ic breast carcinoma diagnosed from 1989 to 1992 were identified retrospectively and clinicopathologic parameters such as age, tumor size, estrogen receptor (ER) and progesterone receptor (PR) status, disease-free survival and overall survival obtained. Paraffin-embedded formalin-fixed tissues were immunostained with bcl-2, Bax and p53 monoclonal antibodies using a standard avidin biotin peroxidase reaction. Stained slides were evaluated by two independent pathologists for staining intensity and percentage of cells staining positively. Cox regression was used for multivariate survival analysis using the clinicopathologic parameters and molecular markers. Chi-square tests were used for frequency tables. RESULTS: Mean patient age was 58 years (range 29 to 79 years) with a median followup of 80 months from time of diagnosis. The most common histopathology was infiltrating ductal carcinoma. Neither bcl-2 nor Bax expression was associated statistically with disease-free or overall survival. Expression of mutant p53 was associated with a significant decrease in both 5-year disease-free survival (70% versus 98%, p

The use of mood stabilizers as plasticity enhancers in the treatment of neuropsychiatric disorders.

Mood disorders have traditionally been conceptualized as neurochemical disorders, but there is now evidence from a variety of sources demonstrating regional reductions in central nervous system (CNS) volume, as well as reductions in the numbers and/or sizes of glia and neurons in discrete brain areas. Although the precise cellular mechanisms underlying these morphometric changes remain to be fully elucidated, the data suggest that severe mood disorders are associated with impairments of structural plasticity and cellular resilience. It is thus noteworthy that lithium and valproate have recently been demonstrated to robustly increase the expression of the cytoprotective protein bcl-2 (an abbreviation for the B-cell lymphoma/leukemia-2 gene) in the CNS in vivo and in cells of human neuronal origin. Lithium and valproate also robustly activate a signaling cascade utilized by endogenous growth factors-the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. Complementary human studies have shown that chronic lithium administration significantly increases gray matter content in a regionally selective manner, suggesting a reversal of illness-related atrophy and an increase in the volume of the neuropil. These unique and unexpected properties of lithium and valproate suggest that they may have broader utility as adjunctive agents in the treatment of a variety of neuropsychiatric disorders associated with cell atrophy or loss. The adjunctive use of these agents-at low doses-may provide the trophic support necessary to restore, enhance, and maintain normal synaptic connectivity, thereby allowing the chemical signal to reinstate the optimal functioning of critical circuits necessary for normal functioning.

Up-regulation of Bcl-2 through hyperbaric pressure transfection of TGF-beta1 ameliorates ischemia-reperfusion injury in rat cardiac allografts.

BACKGROUND: Oxidative stress after ischemia-reperfusion of cardiac allografts leads to activation of cardiomyocytes and production of cytokines. Bcl-2, an inhibitor of the apoptotic pathway, also has strong antioxidant properties. Ischemia-reperfusion injury after transplantation leads to decreased bcl-2 and increased tumor necrosis factor (TNF)-alpha levels. Transforming growth factor (TGF)-beta1 is known to attenuate ischemia-reperfusion injury and inhibits apoptosis of myofibroblasts. We hypothesize that TGF-beta1, prevents bcl-2 cleavage and increased TNF-alpha production. METHODS: Rat PVG donor hearts were heterotopically transplanted into ACI recipients. Donor hearts were procured and assigned to groups: (1) intracoronary TGF-beta1 (200 ng/ml) perfusion and pressure at 78 psi for 45 minutes (n = 4); (2) intracoronary TGF-beta1 perfusion and incubation for 45 minutes without pressure (n = 4), (3) saline perfusion and incubation for 45 minutes without pressure (n = 4). Hearts were procured 4 hours after transplantation and analyzed by reverse transcriptase-polymerase chain reaction for bcl-2 mRNA expression, ELISA for TNF-alpha, and for myeloperoxidase activity (MPO). RESULTS: Bcl-2 decreased in untreated animals (bcl-2:G3PDH ratio = 0.85 +/- 0.73 vs 1.16 +/- 0.11, not significant [NS]), whereas TNF-alpha increased to 669.99 +/- 127.09 vs 276.84 +/- 73.65 pg/mg total protein in controls (p < 0.003). In TGF-beta(1) pressure-treated hearts, bcl-2 was up-regulated (2.49 +/- 0.6 vs 1.16 +/- 0.11, controls, p < 0.005), whereas TNF-alpha was unchanged (396.1 +/- 100.38 vs 276.84 +/- 73.65 pg/mg, NS). Hearts treated with TGF-beta1 and pressure showed significant up-regulation of bcl-2 compared with hearts treated with TGF-beta1 without pressure (2.49 +/- 0.6 vs 1.17 +/- 0.6, p < 0.02). MPO showed no differences. CONCLUSIONS: Bcl-2 is down-regulated and TNF-alpha up-regulated in this model of ischemia-reperfusion injury. Furthermore, TGF-beta1 is linked to this process and ameliorates reperfusion injury by up-regulating bcl-2 and inhibiting TNF-alpha. Therapeutic overexpression of myocardial TGF-beta1 may be clinically useful to control ischemia-reperfusion injury associated with cardiac transplantation.

Apoptosis of airway epithelial cells induced by corticosteroids.

Damage to the airway epithelium is one prominent feature of chronic asthma. Corticosteroids induce apoptosis in inflammatory cells, which in part explains their ability to suppress airway inflammation. However, corticosteroid therapy does not necessarily reverse epithelial damage. We hypothesized that corticosteroids may induce airway epithelial cell apoptosis as one potential explanation for persistent damage. We tested this hypothesis in cultured primary central airway epithelial cells and in the cell line 1HAEo(-). Treatment with dexamethasone, beclomethasone, budesonide, or triamcinolone each elicited a time-dependent and concentration-dependent cell death. This cell death was associated with cleavage of nuclear chromatin, mitochondrial depolarization, cytochrome c extrusion, activation of caspase-9, and expression of phosphatidylserine on the outer cell membrane. Inhibitors of caspase activity blocked apoptotic cell death, as did overexpression of the apoptosis regulators Bcl-2 or Bcl-x(L). We demonstrated that CD95 ligation is not essential for the corticosteroid-induced apoptosis in airway epithelial cells. These data demonstrate that corticosteroids induce apoptotic cell death of airway epithelium. This raises the possibility that at least one of the major components of chronic airway damage in asthma, epithelial shedding and denudation, may in part result from a major therapy for the disease.

The cellular and molecular basis of health benefits of grape seed proanthocyanidin extract.

Red grape seed extract containing proanthocyanidins and other antioxidants are being used as nutritional supplements by many health conscious individuals. The beneficial effects of grape seed proanthocyanidins (GSPE) have been reported, however, little is known about their mechanism(s) of action. One of the beneficial effects of GSPE is chemoprevention of cellular damage. The precise mechanism by which GSPE mediates, chemoprevention is not yet understood. This report addresses this issue. We investigated the mechanisms of actions of GSPE, which ameliorates chemotherapy-induced toxic effects of Idarubicin (Ida) and 4,-hydroxyperoxycyclophosphamide (4-HC) in normal human Chang liver cells. Exposure to GSPE resulted in a significant reduction in apoptosis in response to the cytotoxicity of chemotherapeutic agents. RT-PCR analysis showed a significant increase in the anti-apoptotic gene Bcl-2 and a decrease in the cell cycle associated and proapoptotic genes, c-myc and p53 in cells treated with GSPE. These results suggest that some of the chemopreventive effects of GSPE are mediated by upregulating Bcl-2 and down regulating c-myc and p53 genes.